Antitumor Activities of tRNA-Derived Fragments and tRNA Halves from Non-pathogenic Escherichia coli Strains on Colorectal Cancer and Their Structure-Activity Relationship

ABSTRACT tRNAs purified from non-pathogenic Escherichia coli strains (NPECSs) possess cytotoxic properties on colorectal cancer cells. In the present study, the bioactivity of tRNA halves and tRNA fragments (tRFs) derived from NPECSs are investigated for their anticancer potential. Both the tRNA halves and tRF mimics studied exhibited significant cytotoxicity on colorectal cancer cells, with the latter being more effective, suggesting that tRFs may be important contributors to the bioactivities of tRNAs derived from the gut microbiota. Through high-throughput screening, the EC83 mimic, a double-strand RNA with a 22-nucleotide (nt) 5′-tRF derived from tRNA-Leu(CAA) as an antisense chain, was identified as the one with the highest potency (50% inhibitory concentration [IC50] = 52 nM). Structure-activity investigations revealed that 2′-O-methylation of the ribose of guanosine (Gm) may enhance the cytotoxic effects of the EC83 mimic via increasing the stability of its tertiary structure, which is consistent with the results of in vivo investigations showing that the EC83-M2 mimic (Gm modified) exhibited stronger antitumor activity against both HCT-8 and LoVo xenografts. Consistently, 4-thiouridine modification does not. This provides the first evidence that the bioactivity of tRF mimics would be impacted by chemical modifications. Furthermore, the present study provides the first evidence to suggest that novel tRNA fragments derived from the gut microbiota may possess anticancer properties and have the potential to be potent and selective therapeutic molecules. IMPORTANCE While the gut microbiota has been increasingly recognized to be of vital importance for human health and disease, the current literature shows that there is a lack of attention given to non-pathogenic Escherichia coli strains. Moreover, the biological activities of tRNA fragments (tRFs) derived from bacteria have rarely been investigated. The findings from this study revealed tRFs as a new class of bioactive constituents derived from gut microorganisms, suggesting that studies on biological functional molecules in the intestinal microbiota should not neglect tRFs. Research on tRFs would play an important role in the biological research of gut microorganisms, including bacterium-bacterium interactions, the gut-brain axis, and the gut-liver axis, etc. Furthermore, the guidance on the rational design of tRF therapeutics provided in this study indicates that further investigations should pay more attention to these therapeutics from probiotics. The innovative drug research of tRFs as potent druggable RNA molecules derived from intestinal microorganisms would open a new area in biomedical sciences.


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Reviewer (Comments for the Author):
The MS entitled 'The antitumor activity of tRNA-derived fragments and tRNA halves from non-pathogenic Escherichia coli strain on colorectal cancer and their structure-activity relationship' by Cao et al is rebuttal of work presented to review in June 2020. The MS includes a new mouse experiment which is consistent with the previous data and demonstrates the role of tRSs in vivo. The text has been significantly improved. The quality of the MS for cancer biology is rather limited, but the work is an interesting addition to existing knowledge on tRNA fragments (tRFs) and their implication into cell biology. The authors demonstrated that tRfs from non -pathogenic E.coli are cytotoxic to cell lines and that they present an anti-tumor role in vivo.  Thank you for revising and resubmitting your manuscript. I am pleased to inform you that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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Thank you for submitting your paper to mSystems.    Table S1: Accept Fig. S1: Accept